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Despite the importance of the hERG channel in drug discovery and the sizable number of antagonist molecules discovered, only a few hERG agonists have been discovered. Here we report a novel hERG agonist; SKF-32802 and a structural analog of the agonist NS3623, SB-335573. These were discovered through a similarity search of published hERG agonists. SKF-32802 incorporates an amide linker rather than NS3623's urea, resulting in a compound with a different mechanism of action. We find that both compounds decrease the time constant of open channel kinetics, increase the amplitude of the envelope of tails assay, mildly increased the amplitude of the IV curve, bind the hERG channel in either open or closed states, increase the plateau of the voltage dependence of activation and modulate the effects of the hERG antagonist, quinidine. Neither compound affects inactivation nor deactivation kinetics, a property unique among hERG agonists. Additionally, SKF-32802 induces a leftward shift in the voltage dependence of activation. Our structural models show that both compounds make strong bridging interactions with multiple channel subunits and are stabilized by internal hydrogen bonding similar to NS3623, PD-307243 and RPR26024. While SB-335573 binds in a nearly identical fashion as NS3623, SKF-32802 makes an additional hydrogen bond with neighboring threonine 623. In summary, SB-335573 is a type 4 agonist which increases open channel probability while SKF-32802 is a type 3 agonist which induces a leftward shift in the voltage dependence of activation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia., Competing Interests: This work was supported by GlaxoSmithKline (GSK). EG, BP, ARR, CR, CQ, AS, KW, AR, SS, HQ, HZ, CD, GD, PB, AG, GJ, MFS, MB, GD, NC, NDA, BS, and MTM are employees of GlaxoSmithKline.

Nod-like receptors (NLRs) are cytoplasmic pattern recognition receptors that are promising targets for the development of anti-inflammatory therapeutics. Drug discovery efforts targeting NLRs have been hampered by their inherent tendency to form aggregates making protein generation and the development of screening assays very challenging. Herein we report the results of an HTS screen of NLR family member NLRP1 (NLR family, pyrin domain-containing 1) which was achieved through the large scale generation of recombinant GST-His-Thrombin-NLRP1 protein. The screen led to the identification of a diverse set of ATP competitive inhibitors with micromolar potencies. Activity of these hits was confirmed in a FP binding assay, and two homology models were employed to predict the possible binding mode of the leading series and facilitate further lead-optimization. These results highlight a promising strategy for the identification of inhibitors of NLR family members which are rapidly emerging as key drivers of inflammation in human disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The human, cytosolic enzyme isocitrate dehydrogenase 1 (IDH1) reversibly converts isocitrate to α-ketoglutarate (αKG). Cancer-associated somatic mutations in IDH1 result in a loss of this normal function but a gain in a new or neomorphic ability to convert αKG to the oncometabolite 2-hydroxyglutarate (2HG). To improve our understanding of the basis for this phenomenon, we have conducted a detailed kinetic study of wild-type IDH1 as well as the known 2HG-producing clinical R132H and G97D mutants and mechanistic Y139D and (newly described) G97N mutants. In the reductive direction of the normal reaction (αKG to isocitrate), dead-end inhibition studies suggest that wild-type IDH1 goes through a random sequential mechanism, similar to previous reports on related mammalian IDH enzymes. However, analogous experiments studying the reductive neomorphic reaction (αKG to 2HG) with the mutant forms of IDH1 are more consistent with an ordered sequential mechanism, with NADPH binding before αKG. This result was further confirmed by primary kinetic isotope effects for which saturating with αKG greatly reduced the observed isotope effect on (D)(V/K)NADPH. For the mutant IDH1 enzyme, the change in mechanism was consistently associated with reduced efficiencies in the use of αKG as a substrate and enhanced efficiencies using NADPH as a substrate. We propose that the sum of these kinetic changes allows the mutant IDH1 enzymes to reductively trap αKG directly into 2HG, rather than allowing it to react with carbon dioxide and form isocitrate, as occurs in the wild-type enzyme.

Nucleotide-binding domain leucine-rich repeat proteins (NLRs) play a key role in immunity and disease through their ability to modulate inflammation in response to pathogen-derived and endogenous danger signals. Here, we identify the requirements for activation of NLRP1, an NLR protein associated with a number of human pathologies, including vitiligo, rheumatoid arthritis, and Crohn disease. We demonstrate that NLRP1 activity is dependent upon ASC, which associates with the C-terminal CARD domain of NLRP1. In addition, we show that NLRP1 activity is dependent upon autolytic cleavage at Ser(1213) within the FIIND. Importantly, this post translational event is dependent upon the highly conserved distal residue His(1186). A disease-associated single nucleotide polymorphism near His(1186) and a naturally occurring mRNA splice variant lacking exon 14 differentially affect this autolytic processing and subsequent NLRP1 activity. These results describe key molecular pathways that regulate NLRP1 activity and offer insight on how small sequence variations in NLR genes may influence human disease pathogenesis.

The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss., (© 2011 American Chemical Society)

Heterozygously expressed single-point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively) render these dimeric enzymes capable of producing the novel metabolite α-hydroxyglutarate (αHG). Accumulation of αHG is used as a biomarker for a number of cancer types, helping to identify tumors with similar IDH mutations. With IDH1, it has been shown that one role of the mutation is to increase the rate of conversion from αKG to αHG. To improve our understanding of the function of this mutation, we have detailed the kinetics of the normal (isocitrate to αKG) and neomorphic (αKG to αHG) reactions, as well as the coupled conversion of isocitrate to αHG. We find that the mutant IDH1 is very efficient in this coupled reaction, with the ability to form αHG from isocitrate and NADP(+). The wild type/wild type IDH1 is also able to catalyze this conversion, though it is much more sensitive to concentrations of isocitrate. This difference in behavior can be attributed to the competitive binding between isocitrate and αKG, which is made more favorable for αKG by the neomorphic mutation at arginine 132. Thus, each partial reaction in the heterodimer is functionally isolated from the other. To test whether there is a cooperative effect resulting from the two subunits being in a dimer, we selectively inactivated each subunit with a secondary mutation in the NADP/H binding site. We observed that the remaining, active subunit was unaffected in its associated activity, reinforcing the notion of each subunit being functionally independent. This was further demonstrated using a monomeric form of IDH from Azotobacter vinelandii, which can be shown to gain the same neomorphic reaction when a homologous mutation is introduced into that protein.

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein., (Copyright 2009 Elsevier Ltd. All rights reserved.)

High throughput screening of the corporate compound collection led to the identification of a novel series of 2-amino-9-aryl-3-cyano-4-methyl-7-oxo-6,7,8,9-tetrahydropyrido[2',3':4,5]thieno[2,3-b]pyridine derivatives as selective PR agonists. Initial SAR exploration leading to potent and selective agonists 9 and 11, X-ray crystal structure of 9 bound to PR-LBD and preliminary developability data are described.

We have designed and synthesized a novel series of pyrrolidinones as progesterone receptor partial agonists. Compounds from this series had improved AR selectivity, rat pharmacokinetic properties, and in vivo potency compared to the lead compound. In addition, these compounds had improved selectivity against hERG channel inhibition.

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

A novel series of 1H-indol-1-yl tertiary amine LXR agonists has been designed. Compounds from this series were potent agonists with good rat pharmacokinetic parameters. In addition, the crystal structure of an LXR agonist bound to LXRalpha will be disclosed.

Molecular recognition is the basis of rational drug design, and for this reason it has been extensively studied. However, the process by which a ligand recognizes and binds to its receptor is complex and not well understood. For the case in which the geometries (conformation and alignment) of the ligand and receptor are known from X-ray crystal structure data, the problem is simplified. The receptor-bound conformation and alignment of the ligand is assumed, and those of additional ligands are inferred. For the general case in which the geometries of the ligand(s) and receptor are unknown, no general treatment or solution is available and receptor-ligand geometries must be obtained indirectly from structure-activity studies or synthesis and evaluation of rigid analogs. A general treatment for solving for the receptor-bound geometry of a series of ligands is presented here. Using molecular shape analysis, for ligand description, tensor analysis of N-way arrays by partial least-squares (PLS) regression, and 3-way factor analysis, the receptor-bound geometries of trimethoprim and a series of trimethoprim-like dihydrofolate reductase inhibitors are correctly predicted.